Synthesis and Conformational Studies of Peptidomimetics Containing a New Bifunctional Diketopiperazine Scaffold Acting as a \u3b2-Hairpin Inducer

A practical synthesis of a new bifunctional diketopiperazine (DKP) scaffold 1, formally derived from the cyclization of L-aspartic acid and (S)-2,3-diaminopropionic acid, is reported. DKP-1 bears a carboxylic acid and an amino functionalities in a cis relationship, which have been used to grow peptide sequences. Tetra-, penta-, and hexapeptidomimetic sequences were prepared by solution-phase peptide synthesis (Boc strategy). Conformational analysis of these derivatives was carried out by a combination of 1H NMR spectroscopy, IR spectroscopy, CD spectroscopy, and computer modeling, and reveals the formation of beta-hairpin mimics involving 10-membered and 18-membered H-bonded rings and a reverse turn of the growing peptide chain

A fragment-based virtual screening approach to identify e-cadherin lingands

Cadherins are calcium-dependent cell-cell adhesion proteins which are overexpressed in several solid tumors [1]. They contain an extracellular region consisting of five immunoglobulin-like domains that extend from the cell surface. Recent crystal structures have shown that classical cadherins dimerize through a \u2018strand-swap\u2019 trans-adhesive interface involving the N-terminal EC1 domains of two cadherins on adjacent cells [2, 3]. Despite a growing interest in the field, the rational design of small ligands targeting cadherins is still in a very early stage. Recently, our group set up a docking protocol (Glide v 5.7) to rationally design peptidomimetic ligands mimicking the N- and E-cadherin adhesive homodimer interface. Accordingly, the first mimics based on the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 (DWVI) of the N-terminal adhesion arm were achieved and proved to inhibit the adhesion of epithelial ovarian cancer cells with millimolar potency [4]. Herein, a fragment-based virtual screening approach was applied to identify novel chemical entries targeting the DWVI binding site. Commercially available Maybridge and Life chemicals collections were used. The most promising fragments identified by the docking calculations were purchased and their binding to E-cadherin was evaluated by means of STD (Saturation Transfer Difference) NMR experiments. Acknowledgements: We thank MIUR (PRIN 2015 project 20157WW5EH) for financial support. ____ [1] G. Berx, F. van Roy, Cold Spring Harbor Perspectives in Biology 2009, 1, a003129. [2] D. Leckband, S. Sivasankar, Curr. Opin. Cell Biol. 2012, 24, 620-627. [3] J. Vendome, K. Felsovalyi, H. Song, Z. Yang, X. Jin, J. Brasch, O. J. Harrison, G. Ahlsen, F. Bahna, A. Kaczynska, P. S. Katsamba, D. Edmond, W. L. Hubbell, L. Shapiro, B. Honig, PNAS 2014, 111, E4175-E4184. [4] F. Doro, C. Colombo, C. Alberti, D. Arosio, L. Belvisi, C. Casagrande, R. Fanelli, L. Manzoni, E. Parisini, U. Piarulli, E. Luison, M. Figini, A. Tomassetti, M. Civera, Org. Biomol. Chem. 2015, 13, 2570-2573

Restless Legs Syndrome: Known Knowns and Known Unknowns

Although restless legs syndrome (RLS) is a common neurological disorder, it remains poorly understood from both clinical and pathophysiological perspectives. RLS is classified among sleep-related movement disorders, namely, conditions characterized by simple, often stereotyped movements occurring during sleep. However, several clinical, neurophysiological and neuroimaging observations question this view. The aim of the present review is to summarize and query some of the current concepts (known knowns) and to identify open questions (known unknowns) on RLS pathophysiology. Based on several lines of evidence, we propose that RLS should be viewed as a disorder of sensorimotor interaction with a typical circadian pattern of occurrence, possibly arising from neurochemical dysfunction and abnormal excitability in different brain structures

Side chain effect in the modulation of αvβ3/α5β1 integrin activity via clickable isoxazoline-RGDmimetics: development of molecular delivery systems

View references (57) Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose \u3b1v\u3b23 and \u3b15\u3b21 integrins\u2019 potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as \u201cshuttles\u201d for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards \u3b15\u3b21 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated \u3b15\u3b21 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application

A combined fragment-based virtual screening and STD-NMR approach for the identification of E-cadherin ligands

Cadherins promote cell-cell adhesion by forming homophilic interactions via their N-terminal extracellular domains. Hence, they have broad-ranging physiological effects on tissue organization and homeostasis. When dysregulated, cadherins contribute to different aspects of cancer progression and metastasis; therefore, targeting the cadherin adhesive interface with small-molecule antagonists is expected to have potential therapeutic and diagnostic value. Here, we used molecular docking simulations to evaluate the propensity of three different libraries of commercially available drug-like fragments (nearly 18,000 compounds) to accommodate into the Trp2 binding pocket of E-cadherin, a crucial site for the orchestration of the protein's dimerization mechanism. Top-ranked fragments featuring five different aromatic chemotypes were expanded by means of a similarity search on the PubChem database (Tanimoto index >90%). Of this set, seven fragments containing an aromatic scaffold linked to an aliphatic chain bearing at least one amine group were finally selected for further analysis. Ligand-based NMR data (Saturation Transfer Difference, STD) and molecular dynamics simulations suggest that these fragments can bind E-cadherin mostly through their aromatic moiety, while their aliphatic portions may also diversely engage with the mobile regions of the binding site. A tetrahydro-β-carboline scaffold functionalized with an ethylamine emerged as the most promising fragment

Investigating the interaction of peptidomimetic ligands with e-cadherin using NMR and computational studies

Classical cadherins are versatile calcium-dependent cell\u2013cell adhesion proteins, differentially and specifically expressed in different tissues. Cadherins form homophilic cell\u2013cell interactions by forming dimers between the N-terminal extracellular domains of two cadherins on adjacent cells. Cadherins are known to play a key role in important physiological processes, such as tissue morphogenesis and stability, as well as in the immune system regulation [1]. Over the past 20 years,the expression and/or the dysregulation of several cadherins have been shown to correlate with tumor progression [2]. Thus, cadherins are becoming valuable diagnostic indicators as well as potential therapeutic targets.Recently, our group set up a docking protocol to rationally design small peptidomimetic ligands mimicking the N- and E-cadherin adhesive homodimer interface. Accordingly, the first mimics based on the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 (DWVI) of the N-terminal EC1 domain were achieved (by replacing the central dipeptide Trp2-Val3 with several scaffolds developed in our laboratories) and proved to inhibit adhesion of epithelial ovarian cancer cells with millimolar potency [3]. Molecular Dynamics (MD) simulations were performed starting form the most representative docking poses to discriminate between the stable and unstable docked poses and to equilibrate the system to achieve a stable conformation. MD trajectories have been analyzed according to the experimental information on ligand-cadherin interaction obtained by STD (Saturation Transfer Difference) NMR experiments in the presence of EC1-EC2 construct of the epithelial E-cadherin. NMR data and MD simulations suggest a highly dynamic behavior of both the ligand and the protein and prompt towards an integrated computational and experimental approach to design new small peptidomimetic molecules able to interfere efficiently with cadherin-mediated cell-cell adhesion. Acknowledgements: we gratefully acknowledge Ministero dell\u2019Universit\ue0 e della Ricerca for financial support (FIRB project RBFR088ITV). References 1) D. Leckband, S. Sivasankar, Curr. Opin. Cell Biol. 2012, 24, 620. 2) G. Berx, F. van Roy, Cold Spring Harbor Perspectives in Biology 2009, 1, a003129. 3) F. Doro, C. Colombo, C. Alberti, D. Arosio, L. Belvisi, C. Casagrande, R. Fanelli, L. Manzoni, E. Parisini, U. Piarulli, E. Luison, M. Figini, A. Tomassetti, M. Civera, Org. Biomol. Chem. 2015, 13, 2570

β-Glucuronidase triggers extracellular MMAE release from an integrin-targeted conjugate

A non-internalizing \u3b1v\u3b23 integrin ligand was conjugated to the anticancer drug MMAE through a \u3b2-glucuronidase-responsive linker. In the presence of \u3b2-glucuronidase, only the conjugate bearing a PEG4 spacer inhibited the proliferation of integrin-expressing cancer cells at low nanomolar concentrations, indicating important structural requirements for the efficacy of these therapeutics

Cyclic RGD peptidomimetics containing bifunctional diketopiperazine scaffolds as new potent integrin ligands

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1-DKP-7) or glutamic acid (DKP-8) and feature an amine and a carboxylic acid functional group. The scaffolds differ in the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogen atoms. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins \u3b1 v\u3b2 3 and \u3b1 v\u3b2 5, which are involved in tumor angiogenesis. Nanomolar IC 50 values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2-DKP-8). Conformational studies of the cyclic RGD peptidomimetics by 1H NMR spectroscopy experiments (VT-NMR and NOESY spectroscopy) in aqueous solution and Monte Carlo/Stochastic Dynamics (MC/SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [C\u3b2(Arg)-C\u3b2(Asp) average distance 658.8 \uc5]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin \u3b1 v\u3b2 3 complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex. Copyright \ua9 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

New Insights into the Molecular Mechanism of E-Cadherin-Mediated Cell Adhesion by Free Energy Calculations

Three-dimensional domain swapping is an important mode of protein association leading to the formation of stable dimers. Monomers associating via this mechanism mutually exchange a domain to form a homodimer. Classical cadherins, an increasingly important target for anticancer therapy, use domain swapping to mediate cell adhesion. However, despite its importance, the molecular mechanism of domain swapping is still debated. Here, we study the conformational changes that lead to activation and dimerization via domain swapping of E-cadherin. Using state-of-the-art enhanced sampling atomistic simulations, we reconstruct its conformational free energy landscape, obtaining the free energy profile connecting the inactive and active form. Our simulations predict that the E-cadherin monomer populates the open and closed forms almost equally, which is in agreement with the proposed “selected fit” mechanism in which monomers in an active conformational state bind to form a homodimer, analogous to the conformational selection mechanism often observed in ligand–target binding. Moreover, we find that the open state population is increased in the presence of calcium ions at the extracellular boundary, suggesting their possible role as allosteric activators of the conformational change

The effect of stimulation frequency on transcranial evoked potentials

Introduction - Transcranial magnetic stimulation-evoked electroencephalography potentials (TEPs) have been used to study motor cortical excitability in healthy subjects and several neurological conditions. However, optimal recording parameters for TEPs are still debated. Stimulation rates could affect TEP amplitude due to plasticity effects, thus confounding the assessment of cortical excitability. We tested whether short interpulse intervals (IPIs) affect TEP amplitude.Methods - We investigated possible changes in TEP amplitude and global mean field amplitude (GMFA) obtained with stimulation of the primary motor cortex at IPIs of 1.1-1.4 s in a group of healthy subjects.Results - We found no differences in TEP amplitude or GMFA between the first, second and last third of trials.Discussion - Short IPIs do not affect TEP size and can be used without the risk of confounding effects due to short-term plasticity